Musculoskeletal diseases affect nearly 25% of the American population and is expected to increase with the growing aging population. Furthermore, bone fractures represent a majority of all traumas that take place on the battlefield. Traditional treatment options for these diseases employ autografts and allografts, tissue harvested from one's own bone or donated human cadaver bone, respectively. However issues with tissue scarcity, donor site morbidity, subsequent invasive surgeries in the case of autografts; and possible donor transmitted diseases in the case of allografts makes these treatment options less than ideal. Moreover, processed acellular allografts have limited success in the healing because, although they provide a mechanically compatible scaffold to fill a bone void, these grafts lack viable biological components such as growth factors and cells to initiate osteoinduction, which is the process of initiating the induction of undifferentiated cells towards the osteogenic lineage.
Recent advances in fields of material science, scaffold fabrication and recombinant DNA technology have provided engineers with tools to develop tissue engineered scaffolds to regenerate bone tissue. The desired outcome of tissue engineered bone grafts is the replacement of the graft with new bone growth, therefore an ideal tissue engineered bone graft should be osteoinductive, bioresorbable, and mechanically compatible to host bone.
Bone is a composite tissue comprised of organic and inorganic phases, various cell types, and growth factors. On the nanoscopic level, the extracellular matrix (ECM) of bone consists mainly of an organic phase in the form of fibrillar Type I collagen and an inorganic phase in the form of hydroxyapatite [HA, Ca10(PO4)6(OH)2] particles. The ECM provides mechanical and structural support for surrounding cell types such as osteoblasts, osteocytes and osteoclasts. The precursors of these cell types are osteoprogenitors which originate from mesenchymal stem cells (MSCs). The ECM also serves as a reservoir for growth factors.
Growth factors are signaling proteins that direct cellular proliferation, differentiation, angiogenesis, apoptosis, and even de-differentiation. Growth factors are bound in their latent form to the extracellular matrix (ECM) to sulfated glycosaminoglycans: heparin or heparan sulfate. Upon release from the ECM, growth factors work synergistically and antagonistically in a coordinated, temporal manner to direct the functions of bone regeneration.
Due to proteolytic degradation, growth factors have short half-lives, which is the time to deactivate half of its bioactivity. Therefore, the systemic delivery of growth factors during fracture healing would be ineffective. Local delivery of the growth factor at the site of target is a more effective means of growth factor administration. Recent advances in the development of drug delivery vehicles such as microspheres, hydrogels, and electrospun scaffolds have been promising in the local administration of growth factors.
Electrospinning has been recently utilized in the field of tissue engineering as a scaffold fabrication technique to prepare non-woven scaffolds with fiber diameters on the order of nanometers to microns. The high surface area-volume ratio of the fibers generated in the electrospinning process, makes it an ideal vehicle for various drug delivery applications. The main challenge in the administration of growth factors from electrospun scaffolds is preserving the bioactivity of the incorporated growth factor.
The growth factor can be incorporated at various times in relation to the electrospun composite of the invention, including pre-electrospun incorporation, absorption oro post electrospun incorporation. Pre-electrospun methods include those known in the art such as co-axial electrospinning, emulsion electrospinning and other alternative methods such as electrospinning growth factor encapsulated microspheres, and hydrophobic ion pairing. Post-electrospun growth factor incorporation methods include non-covalent adsorption and growth factor immobilization.
There are various tissue engineering applications for growth factor incorporated electrospun scaffolds such as wound healing, neural regeneration, and musculoskeletal and orthopedic applications.
There has been considerable research in developing growth factor incorporated electrospun scaffolds for the regeneration of tissue from the musculoskeletal system (bone, cartilage, skeletal muscle, etc.) with MSCs which under specific induction conditions can differentiate into osteoblasts, chondrocytes, adipocytes as well as other cell types.
BMP-2 incorporation in electrospun scaffolds is of interest in the osteogenic differentiation of MSCs. Human MSC gene expression of osteogenic markers Alkaline Phosphatase (AP) and Osteocalcin (OC) were upregulated on BMP-2 incorporated electrospun scaffolds compared to PLLA scaffolds without BMP-2.
As known in the art, BMP-2 is integral in mid-to-late stage osteogenic differentiation. Therefore, the release of bioactive BMP-2 is most effective when administered in a sustained manner. Electrospun scaffolds, as known in the art, have been prepared by co-axial electrospinning in which the core solution consisted of BMP-2 incorporated in PEO and the shell solution consisted of a blend of PCL and various concentrations of PEG to manipulate the release rate of BMP-2. Overall, AP activity was higher in hMSCs on scaffolds incorporated with BMP-2 compared to scaffolds without BMP-2. Furthermore, scaffolds prepared with the slow releasing BMP 2 formulation induced higher AP activity compared to scaffolds prepared with a fast releasing BMP-2 formulation. The in vivo analysis showed that de novo bone formation in cranial defects was enhanced with the BMP-2 incorporated scaffolds, with the slow releasing BMP-2 scaffold formulation inducing more bone formation compared to the fast releasing BMP-2 scaffold formulation.
Most growth factor incorporated electrospun scaffolds are comprised of polymers. The addition of a ceramic component to polymers would be an ideal model for bone regeneration because it would reflect the composition of bone in vivo. Investigation in the development of growth factor incorporated polymer/ceramic composite electrospun scaffolds is limited. BMP-2 incorporated PLGA/Hydroxyapatite (HA) composites prepared by emulsion electrospinning and non-covalent adsorption has been investigated.
Characterization of the release kinetics of BMP-2 from electrospun scaffolds revealed a high burst release of BMP-2 from the scaffold prepared by non-covalent adsorption. Furthermore, the effect of different concentrations of HA on the release of BMP-2 was investigated and it was found that increasing the concentration of HA in the composite from 5% (w/w) to 10% (w/w) led to a higher burst release which was thought to be attributed to the hydrophilicity of HA. Human MSCs attachment and viability was highest in preelectrospun BMP-2 incorporated scaffolds prepared with 10% (w/w) HA compared to pre-electrospun BMP-2 incorporated with 5% (w/w) HA, as well as post-electrospun BMP-2 incorporated scaffolds and scaffolds without BMP-2. Scaffolds prepared with BMP-2 improved the healing of critical sized defects in mice tibia.
The effect of BMP-2 incorporated in electrospun composites prepared with a combination of silk, PEO, HA, and BMP-2 by emulsion electrospinning, on the proliferation and osteogenic differentiation of hMSCs has also been investigated. Human MSCs seeded on the BMP-2 incorporated silk/PEO/HA composite had increased calcium deposition as well as higher BMP-2 gene expression compared to scaffolds prepared by the other formulations. This indicates that the PEO and HA enhanced the bioactivity of BMP-2 to initiate osteogenic differentiation of human MSCs.
Hematopoietic stem cells (HSCs) reside in adult bone marrow close to the endosteum. Under either stochastic or deterministic conditions, HSCs differentiate into the various blood cells such as neutrophils, monocytes/macrophages, basophils, eosinophils, erythrocytes, platelets, mast cells, dendritic cells, B and T lymphocytes. Platelets are anucleur cellular fragments, derived from the fragmentation of megakaryocytes. During fracture healing, thrombin activation initiates platelets to release Platelet Derived Growth Factor (PDGF) from the α-granules in its cytoplasm. PDGF is a 25 kDa dimeric glycoprotein that consists of two polypeptide chains linked by a disulfide bond. There are a total of four different polypeptide chains: A, B, C, D that form five different isomers of PDGF: PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and PDGFAB. MSCs and osteoblasts express PDGF receptor-β (PDGFR-β) during fracture healing which bind to the PDGF-BB isoform, which activates a signal transduction pathway that induces cell proliferation, chemotaxis and the upregulation of certain osteogenic markers.
BMPs are members of the transforming growth factor-β (TGF-β) family. The administration of BMP-2, a 26 kDa homodimeric glycoprotein, induces ectopic bone formation and has been implicated in embryonic limb bud formation and bone fracture healing. During fracture healing, BMPs are released from the ECM and bind to BMP receptors (BMPR-I and BMPR-II) on MSCs. This initiates a signal transduction cascade involving the phosphorylation of Smad proteins: Smad-1, Smad-5 and Smad-8, which form a complex with Smad-4 in the nucleus, activating gene expression for proliferation and osteogenic differentiation. BMP-2 is integral throughout the duration of osteogenic differentiation of hMSCs, and has been specifically expressed in the maturation phase of osteoblasts.
However, there remains a need for developing a reliable system for administering growth factors in a manner advantageous to affect bone repair. Further, new methods for growth factor bioactivity preservation must be further developed and investigated, especially with emphasis on the large scale up for possible medical device implementation.